Detection of antibodies specific to various sources of infection (pathogens) such as bacteria and viruses, which may occur in body fluids, is a useful indirect means for the diagnosis of an infection. Therefore, immunological assay techniques and devices designed to detect an antibody by utilizing a pathogen or a component of the pathogen as an assay antigen have heretofore been used in a broad field of diagnosis.
Such an immunoassay method using a pathogen or a component thereof as an assay antigen is advantageous in that the necessary assay system can be easily established but is not fully satisfactory in sensitivity and specificity, thus leaving room for improvement.
As an immunoassay device for use in such immunological assays, there can be mentioned a strip of porous material on which a binding assay (antigen-antibody reaction) is carried out. An assay device of this type takes advantage of the capillary property of a porous substrate, that is to say a body fluid applied to one end of a porous strip migrates toward the other end. Thus, when a test sample (liquid) containing a substance to be assayed is applied to one end of the strip carrying various reagents disposed successively in strategical positions, the sample migrates by capillary action along the strip and encounters those reagents in said positions in succession to undergo reactions. The existence of the substance to be assayed can be confirmed and its amount be determined by detecting a signal from the detectable label included in the ligand-receptor coupling system.
The immunoassay technique utilizing the above principle is often called immunocapillary assay or imunochromatographic assay, and has been described in WO No.87/02774, EP No.0306772 and other publications. As to modifications of the technique, the inventions described in Japanese Unexamined Patent Publication NO.63865/1989, Japanese Unexamined Patent Publication NO.299464/1989 and Japanese Unexamined Patent Publication NO.167497/1994 can be mentioned.
The above-mentioned device is advantageous in that no specific instrument is required for determination and the assay can be completed easily and within a short time but have room for improvement in sensitivity and specificity.
In addition, because the device performs one test only, a negative or positive control sample cannot be concurrently determined, with the consequent disadvantage that it is impossible to judge whether the result is a reliable data generated by the proper determination.
Generally speaking, urine and saliva, among body fluids, are favored as clinical test samples because its collection requires no invasive procedure and is easy and safe as compared with blood.
However, it is usual that the concentrations of antibodies present in such samples are extremely low, for example of the order of one-thousandth to one-ten thousandth of the concentrations in blood. In addition, urine samples collected from subjects who have taken large quantities of water are extremely thin, with the result that a large variation is inevitable in antibody titer among samples.
In such cases, with the conventional assay device described above, the test will be negative when the sample is too thin to detect an antibody, so that the problem arises that the case of “true negative” cannot be differentiated from the case of “negative (false negative)” occasioned by the low concentration of the sample.
Furthermore, when samples lean in antibodies are to be tested, a highly sensitive assay system is required but in that case there is the problem that byproducts formed by nonspecific reactions due to contaminants in the samples are liable to be simultaneously detected to give false positive results.
Therefore, an antibody assay system insuring sufficiently high detection sensitivity even when such body fluids as urine and saliva are used as samples, that is to say a reliable assay system contributory to reduced chances for false negative and false positive tests because of high specificity, is required.
The first object of the present invention is to provide an antibody assay technology (antibody assay method and antibody assay device) which is capable of detecting antibodies against sources of infection occurring in test samples such as body fluids with high sensitivity and high specificity.
The second object of the present invention is to provide an antibody assay method which enables determinations with high accuracy through suppression of “false positive” reactions arising from contaminants in samples even when the samples are those of urine or other body fluid which are comparatively lean in the target antibody.
The third object of the present invention is to provide an antibody assay method as an improvement in immunocapillary assay or immunochromatographic assay, by which the existence and amount of the target antibody as the object of detection in a sample can be accurately determined with a clear demarcation between a “false negative reaction” arising from the nature of the sample and a “true negative” reaction.